This project concerns in-depth studies of the mechanism and function of the release of platelet fibrinogen. The objectives are: 1) to investigate the association of fibrinogen and fibrinogen-related molecules on platelet surface membrane after thrombin treatment; 2) to establish the specific pattern of the release of platelet fibrinogen induced by plasmin; and 3) to develop improved methods for subcellular fractionation of platelets to obtain fibrinogen granules. Specific association of fibrinogen and fibrinogen-related molecules on the surface of thrombin-treated platelets will be investigated 1) by measuring the binding of 125I-antifibrinogen Fab fragments to thrombin-treated platelets and membranes, 2) by determining binding of 125I-fibrinogen to thrombin-treated platelets in the presence of specific inhibitors of ADP-induced aggregation, and 3) by solubilization of surface-bound proteins by digestion with plasmin, trypsin and chymotrypsin or by treatment with 0.5-3.0 M NaCl solution. Solubilized fibrinogen and fibrin in supernatants and bound proteins in platelet pellets and membranes (after lysis) will be extracted by immunoabsorption using antifibrinogen-Sepharose and analyzed by SDS-PAGE. To establish the specific pattern of the plasmin-induced release of platelet fibrinogen, the dose response and time course of the release of fibrinogen and other marker substances for dense granules, alpha-granules and acid hydrolases, will be studied. The effect of "release inhibitors" on fibrinogen secretion will be determined. Changes in platelet subpopulations, membrane proteins and organelle distribution (in fractionation studies) and functions (measured by thrombin and fibrinogen binding and coagulant activities) will by determined after the secretion of fibrinogen from platelets. Further separation and purification of fibrinogen granules from other platelet organelles will be achieved by using linear isosmotic gradient centrifugation and by polymer, aqueous two-phase separation techniques.